Hydrogen has enormous potential to serve as a non-polluting fuel, thereby alleviating the environmental and political concerns associated with fossil energy utilization.
Among the most efficient H2-generating catalysts known are the [FeFe]-hydrogenase enzymes found in numerous microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. The use of Chlamydomonas reinhardtii, also known as green algae, to produce hydrogen from water has been recognized for more than 60 years. The reaction that produces hydrogen is catalyzed by the reversible hydrogenase, an enzyme that is induced in the cells after exposure to a short period of anaerobiosis. This activity is rapidly lost as soon as light is turned on, due to immediate inactivation of the reversible hydrogenase by photosynthetically generated O2 
Ghirardi et al. in Biological Systems For Hydrogen Photoproduction (FY 2004 Progress Report) disclose a method for generating algal hydrogenase mutants with higher O2 tolerance to function with aerobic H2 production systems, which further optimize H2 photoproduction using an algal production system. It generates a recombinant alga expressing an [FeFe]hydrogenase that displays increased tolerance to O2 due to closure of the pathways by which O2 accesses the catalytic site of the enzyme.
T. Happe et al. in Differential Regulation Of The Fe-hydrogenase During Anaerobic Adaptation In The Green Alga Chlamydomonas reinhardtii Eur. J. Biochem. 269, 1022-1032 (2002) disclose using the suppression subtractive hybridization (SSH) approach, wherein the differential expression of genes under anaerobiosis was analyzed. A PCR fragment with similarity to the genes of bacterial Fe-hydrogenases was isolated and used to screen an anaerobic cDNA expression library of C. reinhardtii. The cDNA sequence of HydA contains a 1494-bp ORF encoding a protein with an apparent molecular mass of 53.1 kDa. The transcription of the hydrogenase gene is very rapidly induced during anaerobic adaptation of the cells. The deduced amino-acid sequence corresponds to two polypeptide sequences determined by sequence analysis of the isolated native protein. The Fe-hydrogenase contains a short transit peptide of 56 amino acids, which routes the hydrogenase to the chloroplast stroma. The isolated protein belongs to the class of Fe-hydrogenases. All four cysteine residues and 12 other amino acids, which are strictly conserved in the active site (H-cluster) of Fe-hydrogenases, have been identified. The N-terminus of the C. reinhardtii protein is markedly truncated compared to other non algal Fe-hydrogenases. Further conserved cysteines that coordinate additional Fe—S-cluster in other Fe-hydrogenases are missing. Ferredoxin PetF, the natural electron donor, links the hydrogenase from C. reinhardtii to the photosynthetic electron transport chain. The hydrogenase enables the survival of the green algae under anaerobic conditions by transferring the electrons from reducing equivalents to the enzyme.
Isolation and characterization of a second [FeFe]-hydrogenase gene from the green alga, Chlamydomonas reinhardtii, wherein a HydA2 gene which encodes a protein of 505 amino acids that is 74% similar and 68% identical to the known HydA1 hydrogenase from C. reinhardtii. HydA2 contains all the conserved residues and motifs found in the catalytic core of the family of [FeFe]-hydrogenases disclosed by Forestier et al in Expression Of Two [Fe]-Hydrogenases In Chlamydomonas reinhardtii Under Anaerobic Conditions, Eur. J. Biochem. 270, 2750-2758 (2003). It is demonstrated that both the HydA1 and the HydA2 transcripts are expressed upon anaerobic induction, achieved either by neutral gas purging or by sulfur deprivation of the cultures. Further, the expression levels of both transcripts are regulated by incubation conditions, such as the length of anaerobiosis, the readdition of O2, the presence of acetate, and/or the absence of nutrients such as sulfate during growth. Antibodies specific for HydA2 recognized a protein of about 49 kDa in extracts from anaerobically induced C. reinhardtii cells, strongly suggesting that HydA2 encodes for an expressed protein. Homology-based 3D modeling of the HydA2 hydrogenase shows that its catalytic site models well to the known structure of Clostridium pasteurianum CpI, including the H2-gas channel. The major differences between HydA1, HydA2 and CpI are the absence of the N-terminal Fe—S centers and the existence of extra sequences in the algal enzymes.
It is disclosed that Entamoeba histolytica and Spironucleus barkhanus have genes that encode short iron-dependent hydrogenases (Fe-hydrogenases), even though these protists lack hydrogenosomes in Iron-Dependent Hydrogenases of Entamoeba histolytica and Giardia lamblia: Activity of the Recombinant Entamoebic Enzyme and Evidence for Lateral Gene Transfer Biol. Bull. 204: 1-9. (February 2003). A recombinant E. histolytica short Fe-hydrogenase was prepared and its activity is measured in vitro. A Giardia lamblia gene encoding a short Fe-hydrogenase was identified from shotgun genomic sequences, and RT-PCR showed that cultured entamoebas and giardias transcribe short Fe-hydrogenase mRNAs. A second E. histolytica gene, which encoded a long Fe-hydrogenase, was identified from shotgun genomic sequences. Phylogenetic analyses suggested that the short Fe-hydrogenase genes of entamoeba and diplomonads share a common ancestor, while the long Fe-hydrogenase gene of entamoeba appears to have been laterally transferred from a bacterium. These results are discussed in the context of competing ideas for the origins of genes encoding fermentation enzymes of these protists.
U.S. Patent Application No. 2004/02009256 discloses methods and compositions for engineering microbes to generate hydrogen. Some methods of the invention involve recoding of hydrogenase genes followed by subjecting the recoded genes to annealing-based recombination methods. The invention further provides methods of mating organisms that are transformed with recoded and recombined hydrogenase genes with other organisms containing different genome sequences.
A need exists in the art of H2-generating catalysts of [FeFe]-hydrogenase enzymes, which are found in numerous microorganisms (including C. reinhardtii) to identify the genes essential for formation of active algal [FeFe]-hydrogenase enzymes, due to the fact that expression of an algal [FeFe] hydrogenase structural gene without the co-expression of C. reinhardtii genes results in the accumulation of an inactive [FeFe]-hydrogenase.
Further still, a need exists in the art of H2— generating catalysts of [FeFe]-hydrogenase enzymes to provide co-expression of the C. reinhardtii genes and an algal [FeFe] hydrogenase structural gene in E. coli to produce synthesis of an active [FeFe]-hydrogenase in this bacterium, which lacks a native [FeFe]-hydrogenase.
In the art of H2-generating catalysts of [FeFe]-hydrogenase enzymes, there is yet another need to demonstrate and provide a process to over-express active [FeFe]-hydrogenase in a stable, recombinant E. coli system, and to assemble and insert an H-cluster into C. reinhardtii [Fe]-hydrogenase using the C. acetobutylicum HydE, HydF and HydG proteins to accomplish this activation of non-cognate [FeFe]-hydrogenases—and not limit to the [FeFe]-hydrogenase assembly genes from C. acetobutylicum, the structural genes from C. acetobutylicum or C. reinhardtii, or use of E. coli as an expression host, but to accomplish the expression of any [FeFe]-hydrogenase, expressed in any suitable host, using [FeFe]-hydrogenase assembly genes from any suitable organism.